Generation of three-dimensional meat-like tissue from stable pig epiblast stem cells

Cultured meat production has emerged as a breakthrough technology for the global food industry with the potential to reduce challenges associated with environmental sustainability, global public health, animal welfare, and competition for food between humans and animals. The muscle stem cell lines currently used for cultured meat cannot be passaged in vitro for extended periods of time. Here, we develop a directional differentiation system of porcine pre-gastrulation epiblast stem cells (pgEpiSCs) with stable cellular features and achieve serum-free myogenic differentiation of the pgEpiSCs. We show that the pgEpiSCs-derived skeletal muscle progenitor cells and skeletal muscle fibers have typical muscle cell characteristics and display skeletal muscle transcriptional features during myogenic differentiation. Importantly, we establish a three-dimensional differentiation system for shaping cultured tissue by screening plant-based edible scaffolds of non-animal origin, followed by the generation of pgEpiSCs-derived cultured meat. These advances provide a technical approach for the development of cultured meat.

For the 3d tissues, there is again an absence of convincing differentiation data.The supplementary material (Fig. S5) does contain some myosin stainings that look promising in terms of myotube formation, although again these are provided without quantification or adequate controls for comparison.In fact, the tubes that were formed are surprisingly reminiscent of tube formation that is seen in endothelial cells and some mesenchymal cells.S5A likewise seems to show several multinucleated myotubes, but it is hard to assess by eye what proportion of cells are actually fused.Given that this point is fairly central to the author's claim to be producing differentiated meat-like tissue, these data need to be extended, improved and emphasized more heavily in the presentation of the article.
For both 2d and 3d, there are no robustly quantifiable protein measurements, for example by western blot for major muscle proteins, which needs to be addressed.A favourable comparison with traditional meat would not be required for publication, but certainly a strong induction relative to undifferentiated cells would be expected.
The RNAseq and metabolomics data are impressive, but I somewhat question the value of these data (particularly the extent to which they are presented, as two entire figures) when there is no real way to quantify these changes relative to a differentiated sample.The metabolomics data add very little to the central statement of the study.The "crucial pathways",mentioned on line 237 for example requires much more context to be relevant for the story.
The differentiation scheme seems unusually long, given that most myogenic differentiation protocols take place over a matter of days.Some timecourse data to investigate whether this process is really required might be interesting.At a bare minimum, the authors should discuss this point and the potential limitations that it might have for an cultured meat production process based on this system.
It is difficult to assess differentiation efficiency (percentage of cells/clones that participate in differentiation) as only level of differentiation is shown and compared to literature data, not positive controls (e.g.C2C12s) Scaffold: Please show cell adhesion to scaffold material.This point in the introduction is not really the case, as immortalised adult stem cells, such as bovine satellite cells, are now available (Stout et al).Such lines are also available for avian species, such as chicken.
It is rather hard to know what to make of the karyotype analysis from the provided figure(s), given that no quantification nor labelling is provided.
The article and figures need to be thoroughly checked over for English spelling and grammar.F-actin should be spelt with a hyphen.'Cancer hallmarkers' does not make sense as a graph title, and there are many other examples.Some of the graph axes are rather strangely named, for example 'The damage levels of DNA'.
Reviewer #3 (Remarks to the Author): I co-reviewed this manuscript with one of the reviewers who provided the listed reports as part of the Nature Communications initiative to facilitate training in peer review and appropriate recognition for co-reviewers.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): This research highlights the successful development of a differentiation system using pgEpiSCs for CM production.It also emphasizes the potential benefits of CM and the significance of the findings for the future of CM development.All the biological and biomolecular characterizations were in-depth and well-executed.This work also established a 3D differentiation system to shape cultured tissue.The team achieved this by screening plant-based edible scaffolds of non-animal origin, namely sodium alginate (SA) and konjac glucomannan (KGM) composite hydrogel, through a physical ionic crosslinking process.This research effectively addresses the first limitation of cultivated meat, which is cell line development.However, there are some minor issues that need to be addressed before this work is ready for publication.Please see the point-by-point comments below: (1) Which part of the fresh pork was selected for the TPA test?Was it the loin or other parts?R: Thanks for your comment.It is the loin.The porcine loin has an important economic value as an important edible part of the pig, and porcine muscle stem cells (pMuSCs) were isolated from the longest dorsal muscle, which contains porcine loin, so we chose porcine loin for comparison of textural properties with pgEpiSC-derived CM, which is described in the methods of the revised manuscript (as shown in Line 726).
(2) Besides ionic crosslinking, what is the reason behind choosing KGM and SA as the plant-based materials?There are other plant-based materials that also contain protein, such as soy, pea protein hydrolysate, zein, etc.Why did the authors not incorporate these protein-based materials to increase the final product's nutritional value?R: Thanks for your comment and question.Konjac glucomannan (KGM) and sodium alginate (SA) were chosen as plant-based materials for the following reasons: 1) As you pointed out, it was initially based on the cross-linking capabilities of SA and calcium ions 1 , which are particularly advantageous for preparation process simplicity, safety, and cost control and have significant potential for large-scale creation of CM scaffolds.2) Regarding the substance itself, SA is an anionic polymer that occurs naturally and is typically derived from brown seaweed 2 .It has long been extensively used in the food industry due to its abundance, good biocompatibility, low cost, and mild gelation, as well as the structure of the hydrogel it creates, which is comparable to the extracellular matrix of living tissues 3 .3) SA hydrogels alone are difficult to apply directly to the preparation of CM scaffolds due to the lack of mammalian cell integrin adhesion ligands 4 .Furthermore, KGM, a water-soluble polysaccharide, has drawn more attention owing to its favorable biocompatibility, biodegradability, and hydrophilicity, as well as its lack of danger and harmful effects 5 , which may be utilized as a suitable material for scaffolds made from CM.As a result, we chose to combine KGM and SA for the creation of plant-based scaffolds without animal-derived components.
We agree with the suggestion you provided, and we have also made attempts with soy protein isolate (SPI) and zein as additional scaffold production ingredients to increase the final product's nutritional value.However, the results did not match expectations (as stated below, Response Figure 1).We added zein to the ethanol replacement solution since it is alcohol-soluble and substituted soy protein isolate for an appropriate proportion of the original preparation solution.The results demonstrated that the addition of zein was unable to infiltrate through the scaffold and could only form a yellow zein film on the surface (Response Figure 1A, E).With the addition of medium, the culture system became murky, zein precipitated, and the scaffold's structure collapsed, making it difficult to carry out nutrient replenishment and unsuitable for the growth of cells (Response Figure 1B-D, E).
Although the scaffolds were structurally intact at the initial stage of culture after the addition of SPI replacement (Response Figure 1A, F), the scaffolds became soft after further inoculation with C2C12 cells and culture for 24 h (Response Figure 1H).It was difficult to maintain a stable structure for long-term culture, and the results of the live/dead cell staining revealed an increase in the number of dead cells with an increase in the proportion of SPI addition (Response Figure 1G), which requires that further new technological approaches be sought for the incorporation of plant proteins; therefore, we did not add the results in the revised manuscript.
Additionally, plant-based scaffolds (such as texturized soy protein 6,7 , wheat glutenin 8 , peanut wire-drawing protein prolamins 9,10 , or decellularized plants 11 ) have been reported to reduce the dependence on animal-derived components; however, they have poor cell adhesion abilities.The addition of animal-derived thrombin 6 , fibrinogen 12 , collagen 13 and gelatin 14 is required to promote cell adhesion, which is also commonly used as a solution.
In contrast, the advantage of our plant-based scaffolds is that the present scaffold, made utilizing KGM with SA without any animal-derived components, has a good affinity with cells and does not require the inclusion of any animal-derived components to enhance cell adhesion.Of course, we continue to strive harder to improve the nutritional value of the final product by adding nutrients like soy protein during food processing.respectively.F, Appearance of SPI-Ca 2+ -KGM-SA scaffolds after addition of medium.G, Representative images of calcein-AM (green) and PI (red) of C2C12 cultured onto the SPI-Ca 2+ -KGM-SA scaffold for 24 h.Scale bar, 100 μm.The SPI-Ca 2+ -KGM scaffold collapsed structurally after 24 h of cell culture inoculation, and the three leftmost figures show its appearance after collapse.H, Focus images of SPI-Ca 2+ -KGM-SA scaffolds, taken in the following order: scaffolds after ethanol replacement (left); wells after medium addition (middle); scaffolds after C2C12 inoculation and 24-hour incubation (right).R: Thanks for your comment.Since current researches 1,2,3 suggested that F-actin could be utilized to identify myofibers, we used F-actin and DAPI to evaluate the threedimensional differentiation of pgEpiSCs-MCs on 3D plant scaffolds without animal-derived components.According to the suggestion, we performed additional experiments of immunofluorescence staining using Myosin and MF20, and the results showed that pgEpiSCs-MCs had good capacity to differentiate on this plant-based scaffold.This result was added to the revised manuscript (as shown in Supplementary Fig. 12f).How can the authors ensure that they were fully proliferated and differentiated on the scaffolds?R: Thanks for the comment.We performed additional experiments by SEM to confirm that the cells attached to the scaffolds properly and could adhere well to the scaffold.We further repeated the experiment several times with similar results and hypothesized that the cell morphology may be associated with the scaffold material, its permeability to light, or the cell type.Similar results were observed when the paper's authors incubated mouse fibroblasts (L929 cells) with USO-grafted cotton gauze 1 .

Notes:
The figure was cited from references 1 .
Additionally, the proliferation status of the pgEpiSCs-MCs on this plant-based scaffold was also confirmed by counting the number of cells with the nuclear localization of GFP signals on various days (as shown in Supplementary Fig. 12a).The number of cells significantly increased from day 1 to day 10, and the results of immunofluorescence staining (F-actin, Myosin, and MF20) revealed that pgEpiSCs-MCs underwent differentiation and multinucleated myotube formation was observed (as shown in Fig. 5d and Supplementary Fig. 12f).
(5) Could the authors provide zoomed-in SEM images to observe how the cells interact with the scaffolds?R: Thanks a lot for your suggestion.We repeated the cell inoculation experiment to observe how the cells interact with the scaffolds, and this result was added to the revised manuscript (as shown in Supplementary Fig. 12c).The results of zoomed-in SEM images showed that pgEpiSCs-MCs were able to stably attach to the plant-based scaffolds without any animal-derived components.Cell adhesion and stretching could also be clearly seen after cell attachment to reveal a full cell shape.However, the shapes of the plant scaffolds with unattached cells showed smooth surfaces.

Notes:
The interaction of pgEpiSCs-myoblast inoculated for 24 h with the scaffold was observed using SEM.Scale bar, 20 μm.Cells are indicated with red arrows, the scaffold outline without cells attached is marked by blue arrows.Overall this is an impressive piece of work, which spans a range of relevant topic areas in cultured meat, from cell line generation through to tissue formation.Whilst the results presented are certainly interesting, at this point in time, there are some major weaknesses in the study.The primary weakness that needs to be addressed is the demonstration of myogenic differentiation at the protein level, in both 2d and edible 3d constructs.
Please find attached some major and minor comments.
Major comments: (1) The major weakness in this study is the lack of convincing evidence of myogenic differentiation, and a strong overreliance on bulk RNA-based data to try to make this point.
R: Thanks to the reviewer for this comment.First, we succeeded in trying to direct the differentiation of pgEpiSCs into muscle cells (MCs) without the use of serum, and the results are shown in Fig. 2 and its Supplementary figures.Here, we performed transcriptome sequencing to reflect general biological features of the differentiated cells in order to clarify the feasibility of the established serum-free myogenic differentiation system.
The histological data also showed that pgEpiSCs as pluripotent stem cells were differentiating into MCs, and more myogenic differentiation genes were detected.
Meanwhile, we performed RT-PCR to verify the detected genes related to myogenic differentiation and collagen formation in order to confirm the validity of the transcriptome data (as shown in Fig. 3f, g).
In addition, we performed additional experiments to determine the expression of OCT4 (related to pluripotency) and MYH3 (related to myofiber maturation) during the myogenic differentiation of pgEpiSCs by western blot at the protein levels (as shown in Fig. 2h, j).
Furthermore, we performed immunofluorescence staining of MF20, and our results suggested that pgEpiSCs-MCs could indeed form multinucleated myotubes with higher magnifications and quantified the fusion index (as shown in Fig. 2f, g, i).As well, we performed experiments to address your concern about determining whether pgEpiSCs-MCs had endothelial or mesenchymal cell characteristics.Besides, we also analyzed the trends in gene expression associated with myogenic differentiation over time courses and discussed the long period of the differentiation process with the limitations of the CM production process in the revised manuscript.All of these results were added to the revised manuscript.Meanwhile, we attempted experiments with a variety of myofiber-related antibodies (such as MF20, Myosin, TITIN, MyHc, or Dsemin) to identify myofibrillar features in pgEpiSCs-MCs, but commercial antibodies against livestock animal species are scarce and interspecies-specific, resulting in limitations to immunofluorescence or western blot 1 ; therefore, we further illustrated the features of pgEpiSCs-MCs by RT-PCR and histological sequencing technology.
( 2f-j and Supplementary Fig. 5a).These results were added to the revised manuscript.Along with this, we analyzed the genes associated with mesenchymal differentiation (CD90 and CD105) and endothelial differentiation (CD31 and CDH5).We found that the endothelial differentiation-related genes (CD31 and CDH5) were not yet expressed in the myogenic differentiation system of pgEpiSCs, whereas the mesenchymal differentiationrelated genes (CD90 and CD105) were elevated and then decreased.This result was added to the revised manuscript (as shown in Supplementary Fig. 6 and Lines 187-189).
(3) For the 3d tissues, there is again an absence of convincing differentiation data.
The supplementary material (Fig. S5) does contain some myosin stainings that look promising in terms of myotube formation, although again these are provided without quantification or adequate controls for comparison.In fact, the tubes that were formed are surprisingly reminiscent of tube formation that is seen in endothelial cells and some mesenchymal cells.S5A likewise seems to show several multinucleated myotubes, but it is hard to assess by eye what proportion of cells are actually fused.Given that this point is fairly central to the author's claim to be producing differentiated meat-like tissue, these data need to be extended, improved and emphasized more heavily in the presentation of the article.
R: Thanks for your comment.For three-dimensional differentiation, we performed additional experiments to confirm the myogenic differentiation by the assays of western blot and immunofluorescence staining for the expressions of OCT4, MYH3, F-actin, MF20 and Myosin, and quantified the results of the protein assays.The results were added to the revised manuscript (as shown in Fig. 5d and Supplementary Fig. 12f, h, i).In addition, we have provided undifferentiated cells as a negative control (as shown in Fig. 2f) and quantified the fusion index (Fig. 2i).
Additionally, we performed extra experiments to address your concern about determining whether pgEpiSCs-MCs had endothelial or mesenchymal cell characteristics, as shown below (Response Figure 2).pgEpiSCs were directed to differentiate into endothelial cells (ECs) and mesenchymal cell-like cells (MSCLCs) in the absence of serum (Response Figure 2A), using the methods in the previous study [1][2][3][4][5] and the cellular morphology, gene expression, and immunofluorescence staining were analyzed.We found that pgEpiSCs, pgEpiSCs-MCs, pgEpiSCs-MSCLCs, and pgEpiSCs-ECs were significantly different in morphology (Response Figure 2B).The differentiated cells also References: (4) For both 2d and 3d, there are no robustly quantifiable protein measurements, for example by western blot for major muscle proteins, which needs to be addressed.
A favourable comparison with traditional meat would not be required for publication, but certainly a strong induction relative to undifferentiated cells would be expected.
R: Thanks for your suggestion.We agree with the reviewer's comment, and the muscle protein marker (MYH3) and the pluripotency marker (OCT4) were measured by western blot to demonstrate the induction of 2D and 3D at the protein level.The results showed that the expression of MYH3 was significantly increasing in pgEpiSCs-MCs in comparison to undifferentiated cells, which quantified the results of the protein assays in 2D or 3D differentiation.We have added these results to the revised manuscript (as shown in Lines 181-185, 277, Fig. 2h, j and Supplementary Fig. 12h, i).
( R: Thanks to the reviewer for this comment.As in reply to Comment 1, we performed transcriptome and untargeted metabolomic sequencing to reflect general biological features of the differentiated cells in order to clarify the feasibility of the established serumfree myogenic differentiation system.Our results indicated that the serum-free myogenic differentiation of pgEpiSCs is involved in metabolic pathways such as vitamin B6, the pentose phosphate pathway (PPP), mineral absorption, protein digestion and absorption.
1) Vitamin B6, as a key cofactor for various biochemical reactions in basal cellular metabolism, has an important role in muscle production 1,2 .Besides, as a dietary nutrient additive component, it promotes muscle development and regeneration in the organism 2 , which also suggests that the vitamin B6 metabolic pathway plays an important role in the process of muscle development.
2) The PPP is a glucose metabolism process 3 .Myogenic differentiated cells may change their biosynthesis rate, resulting in lower amounts of chemicals linked to glycolysis and the PPP, which implies that myogenic differentiation depends on glycolytic metabolites 4 .
3) Mineral absorption affects myogenic differentiation by influencing Ca 2+ , Mg 2+ , and other ions.Mg 2+ promotes myogenic differentiation in C2C12 and aged MuSCs 5 , Ca 2+ also accelerates myogenic differentiation and myotube formation 6 , which suggests that mineral uptake has an important effect on myogenic differentiation.
4) Protein digestion and absorption are also able to influence myogenic differentiation.
Tryptophan-fed mice could enhance myogenic differentiation and myotube maturation 2,7 , and the tryptophan metabolism was upregulated in late differentiation 8 .Meanwhile, arginine can accelerate myogenic differentiation and myotube formation through synergistic Ca 2+ action 6 .
Although a clear mechanism of action for these pathways has not yet been described, this is sufficient to suggest that myogenic differentiation is associated with these pathways.
In addition, glycolysis plays an important role in myogenic differentiation 3,4 and we further validated the involvement of glycolysis-related genes at the RT-PCR level.In the field of skeletal muscle research and the meat industry, it has been suggested that metabolomics is not only a key technique for characterizing meat composition and investigating biomarkers for quality control but can also be used in a way that will help to optimize the composition of the culture medium during myogenic differentiation 9 .Meanwhile, these data help to understand the metabolic behavior of MCs during serum-free induction and offer a possible technical approach to further optimize the formulation of the medium by subsequently controlling the metabolic level of glucose and other metabolites, which were described in the discussion of the revised manuscript (as shown in Lines 332-337).
Line 175: the corresponding figure says 99.8% Supplementary Fig 9H y-axis labels are missing Supplementary Fig 10D, please provide stress Pa, to provide reference to muscle tissue Line 316.There are several studies in literature proving this wrong Minor comments "Currently accessible cell lines include mesenchymal stem cells and muscle stem cells (MuSCs), which can be amplified only a limited number of times before losing their capacity to differentiate."

Figure 1 :
Preparation and feasibility analysis of Zein/SPI-Ca 2+ -KGM-SA scaffolds.A, Focus diagram of the appearance of Zein/SPI Ca 2+ -KGM-SA scaffolds, where Zein is dissolved in ethanol replacement solution according to the ratio, and SPI is the scaffold added to the KGM-SA mixing solution.B, The types of scaffolds and mixing ratios corresponding to Figs.C, D, F and G. C, Appearance of Zein-Ca 2+ -KGM-SA scaffolds.D, Appearance of Zein-Ca 2+ -KGM-SA scaffolds after addition of medium.E, Focus views of the Zein-Ca 2+ -KGM-SA scaffolds, taken in the order of ethanol replacement, medium addition to the wells, and scaffolds, are shown in the left, middle, and right images,

( 3 )
Why was the confocal experiment only performed with F-Actin and DAPI?Why were other immunofluorescence staining, such as Myogenin, Desmin, or Myosin heavy chain, not included to assess the cells cultured on the 3D scaffold?

( 4 )
Based on the Calcein/PI staining, the cell morphology appears to be very round.

( 6 )
Apart from the amino acid profile provided in the supplementary materials, will the authors be able to provide the total protein content and other nutritional values for the pgEpiSCs-CM sample?R: Thanks for your suggestion.We first measured the amino acid composition of pgEpiSCs-CM to evaluate its nutritional value.Additional experiments were performed on the total protein content and other nutritional values (such as fat content and trace elements) for the pgEpiSCs-CM sample with the aid of your helpful suggestions.The total protein content in pgEpiSCs-CM was around 3.92% compared to the plant-based scaffolds without cell inoculation, and trace elements (such as Zn, Ca, Mg, and K) were found.This result was added to the revised manuscript (as shown in Supplementary

)
In general, the differentiation images provided are of poor quality, and are not quantified in any fashion (for example using fusion index, which is very standard in the field of myogenesis).From the brightfield images, it seems like a lot of cells are aligned, but probably not fused, although it is hard to tell.The fluorescent stainings likewise show little convincing evidence of fusion, even though there is plenty of signal.No undifferentiated controls are provided for comparison, nor is there any time course data showing the process of myogenesis.These data must be substantially improved with higher quality images at higher magnifications.R: Thanks for your comment.We repeated the differentiation experiments to improve the quality of the images and confirmed the multinucleated myotubes fused by immunofluorescence staining, as well as quantified the fusion index by MF20 immunostaining and provided undifferentiated cells as a negative control.The pgEpiSCsderived MCs showed that multiple spindle-shaped nuclei were arranged, suggesting multinucleated myotube formation, which was further confirmed by expressing markers of mature skeletal muscle fibers (SMFs), such as Myosin, MF20, and MYH3 (as shown in Fig. According to the suggestion about time course data showing the process of myogenesis, we collected cell samples every two days for RT-PCR to analyze the expression of relevant genes (as shown below), including key marker genes related to pluripotency (OCT4 and NANOG), paraxial mesoderm differentiation (T and MSGN1), muscle stem cells (PAX7 and MYOD), myogenic maturation (MYOG, MYH3, and MYH11), mesenchymal differentiation (CD90 and CD105), and endothelial differentiation (CD31 and CDH5).The results indicated that the pluripotency genes (OCT4 and NANOG) were downregulated as differentiation advanced and did not significantly change during the later differentiation process, whereas paraxial mesoderm differentiation-related genes (T and MSGN1) and muscle stem cell-related genes (PAX7 and MYOD) showed a tendency for increasing and then decreasing, which also indicated directional myogenic differentiation.Meanwhile, the expression of MYOG, MYH3, and MYH11 fluctuated a little bit at a later stage, but it remained high compared to undifferentiated cells or pgEpiSCs-derived MPCs.
figures) when there is no real way to quantify these changes relative to a differentiated sample.The metabolomics data add very little to the central statement of the study.The "crucial pathways", mentioned on line 237 for example requires much more context to be relevant for the story.

Response Figure 3 :
participate in differentiation) as only level of differentiation is shown and comparedto literature data, not positive controls (e.g.C2C12s).R: Thank you for your comment.Here, we determined the efficiency of pgEpiSCs to differentiate into MPCs.We also performed experiments to evaluate the differentiation effectiveness of pgEpiSCs-MPCs in accordance with your suggestion, utilizing C2C12 as a positive control, and the results were shown below (Response Figure3).Firstly, the results of immunofluorescence suggested that C2C12 expressed MYOD and PAX7 was negatively expressed (Response Figure3A), in contrast to pgEpiSCs-MPCs, which expressed PAX7 and MYOD simultaneously.Next, the proportion of CD31 -CD45 -CD56 + was only 0.14% for C2C12, according to the results of the flow cytometry, whereas the percentage of pgEpiSCs-MPCs was positive up to 99.8% (Response Figure3B).Furthermore, co-expression of PAX7 and MYOD is a characteristic of muscle stem cells (MuSCs), while CD56 is also a marker of MuSCs 1 .These results indicated that pgEpiSCs-MPCs possessed the characteristics of MuSCs, whereas C2C12, as an immortalized myoblast cell line, does not have the characteristics of MuSCs.In addition, we chose to compare the myogenic differentiation efficiency of pluripotent stem cells with that of humans and mice since there are no pluripotent stem cell lines that can be passed on stably for a long time in vitro in livestock species (such as pigs).Comparative analysis of differentiation efficiency of pgEpiSCs-MPCs.A, Immunostaining of PAX7and MYOD in C2C12 and pgEpiSCs-MPCs.Scale bar, 20 μm.B, Flow cytometric analysis of the proportion of CD31 -CD45 -CD56 + cell populations in C2C12 and pgEpiSCs-MPCs.